While isolating phages from natural sample, I got different plaques indicating mixed information. I want to isolate a single phage so started culturing with only 1 plaque. I took out single plaque , mixed it with 1ml buffer and was trying to do plaque count but I am not getting plaques as @ 10^-18 dilutions also show clear lysis.
You do not have a good lawn at all so you would not be able to see any plaques if they existed.
It is hard to diagnose the problem but either the culture you are growing for the lawn was contaminated perhaps with phage and lots of cells are dead, or maybe your top agar was too hot and you killed lots of the plating cells. But that is not a normal looking lawn.
well first do a spot test by serial dilution do it upto 12th dilution then do a Double agar overlay. After that from the DAOL plate, pick a single isolated plaque and do a phage purification, repeat this step three times then proceed
Make sure you run controls. Did you also plate a negative control (only the buffer you used for the dilution series)? I would definitely do that, because it will allow to decide what parts of your experiment are ok. As Michael J. Benedik said, it could be that the bacteria culture is already contaminated. Or maybe the buffer, who knows.
...and because tracking all of this down requires a lot of plates, don't do it that way. Instead, pour your soft agar with bacteria on the plate, but then spot 10 ul of your dilution on the plates. Actually, if you spot 2-5 ul, you can even use an eight-channel pipette and spot 48 samples on one plate. Saves you a lot of time and gives you enough information for the start (including a negative control for free).
Hello thank you all for your suggestions. I have ran control every time and bacteria grows quite well in that plate. That's why I am quite surprise how this is happening. I would try to do spot assay.
Hopefully the spot assay will help, but if you are still having issues please show some other pictures including of the control plate.
What is your host bacterium? Do you grow it from a triple-serial-isolated colony? I agree with Michael & Dominik. Please demonstrate a smooth, uninterrupted lawn of your host first.
-After triple-serial-isolated colony is acquired, confirm host identity. Determine timing of log-phase (media, temperature, shaking rate).
-Grow overnight culture to stationary phase, dilute stationary phase 1:10 in fresh broth & grow into 'determined' log phase.
-Pour log phase culture over 8 fresh TSA (or appropriate media), allow to dry somewhat in sterile hood, until no host runniness is present.
-Refrigerate 4 of the host plates. Do phage SPOT-dilution assays on 2 host plates and incubate alongside 2 host-only (no phage) plates.
-Host only plates should show a smooth turbid lawn. SPOT-dilution plates should show cleared or turbid spots, depending on your phage concentration and/or type.
-If all goes well, you still have 4 ready-to-use host plates in refrigeration.
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